Larvicidal activity of Acacia nilotica extracts against Culex pipiens and their suggested mode of action by molecular simulation docking

Mosquitoes are one of the deadliest and most hazardous animals on Earth, where they transmit several diseases that kill millions of people annually. There is an ongoing search almost everywhere in the world for more effective and contemporary ways to control mosquitoes other than pesticides. Phytochemicals are affordable, biodegradable biological agents that specialize in eliminating pests that represent a risk to public health. The effectiveness of Acacia nilotica methanol and aqueous leaf extracts against 4th instar larvae was evaluated. The results revealed that the methanol extract of A. nilotica had a noticeable influence on the mortality rate of mosquito larvae, especially at high concentrations. Not only did the mortality rate rise significantly, but the hatching of the mosquito eggs was potentially suppressed.Terpenes, fatty acids, esters, glycosides, pyrrolidine alkane, piperazine, and phenols were the most prevalent components in the methanol extract, while the aqueous extract of A. nilotica exclusively showed the presence of fatty acids. The insecticidal susceptibility tests of both aqueous and alcoholic extract of A. nilotica confirmed that the Acacia plant could serves as a secure and efficient substitute for chemical pesticides because of its promising effect on killing larvae and egg hatching delaying addition to their safety as one of the natural pesticides. Molecular docking study was performed using one of the crucial and life-controlling protein targets, fatty acid binding protein (FABP) and the most active ingredients as testing ligands to describe their binding ability. Most of the structurally related compounds to the co-crystallized ligand, OLA, like hexadecanoic acid furnished high binding affinity to the target protein with very strong and stable intermolecular hydrogen bonding and this is quite similar to OLA itself. Some other structural non-related compounds revealed extraordinarily strong binding abilities like Methoxy phenyl piperazine. Most of the binding reactivities of the majortested structures are due to high structure similarity between the positive control, OLA, and tested compounds. Such structure similarity reinforced with the binding abilities of some detected compounds in the A. nilotica extract could present a reasonable interpretation for its insecticidal activity via deactivating the FABP protein. The FABP4 enzyme inhibition activity was assessed for of both methanolic and aqueous of acacia plant extract and the inhibition results of methanol extract depicted noticeable potency if compared to orlistat, with half-maximal inhibitory concentration (IC50) of 0.681, and 0.535 µg/ml, respectively.


Plant materials and analysis
Plant collection and extraction Leaves of Acacia plants, Acacia nilotica (Fabaceae) were collected from different areas in Qalyubiya Governorate, Egypt, during August-September 2022.Plants were identified in the Flora and Phytotaxonomic Section of the Agricultural Research Centre in Giza, Egypt.The plant components were shade-air-dried at room temperature for seven days until the plant was thoroughly dried and the dry weight contracted.To keep the dried tissues free from moisture, they were pulverized in an electric mixer made of stainless steel and placed within airtight containers.To make the methanol extract, 20 g of dry plant materials were soaked in 100-150 ml of methanol in a conical flask (cap.250 ml) at room temperature 27 ± 2 °C.Another 20 g of plant components were mixed with warm water (60 °C) at room temperature.After 48 h, the solutions were filtered via a Buchner funnel using Whatman No. 1 filter paper.A rotary evaporator was used to concentrate the extract for 3-5 h at room temperature (28 °C) and solid product then stored at 4 °C until used 46 .

Mosquito larvicidal assay
Culex pipiens rearing Larvae of Cx. pipiens were raised in the insectary at the Department of Entomology, Faculty of Science, Benha University, Egypt, for 12 generations, where they were kept at 27 ± 2 °C and 75 ± 5% relative humidity during a light/dark photoperiod of 12:12 h.The larvae were fed a 3:1 ratio of ground bread to fish meal (Tetramin).After being transferred from the enamel pans to a cup of dechlorinated water, the pupae were placed in screened cages of 35 × 35x40 cm, from which the adults finally emerged.The adult mosquito colony received a 10% sugar solution, while the females were periodically fed blood by a hamster rat.Two developmental stages, larvae, and pupae were consistently accessible for the testing and maintained in the same lab.

Larvicidal activity
Under laboratory circumstances, the plant leaf methanol, and aqueous extracts of A. nilotica were tested against the 4th larval instar of Cx. pipiens (No. = 20 larvae).The fourth larval instar was exposed to doses of 62.5, 125, 250, 500, 1000, and 1500 ppm (1 g/1000 ml distilled water).Twenty larvae were introduced to a glass beaker holding 250 ml of distilled water for each concentration.Each concentration received three replicates.Temephos was employed at a rate of 0.25 ppm as a positive control according to WHO 47 standard concentration within a similar volume of test beaker 48 .Mortality rates were obtained 24 h, 48 h, and 72 h after the first exposure and post-treatments (PT) 12 .No food was provided to the larvae during the experimental period.

Oviposition assay
To analyze the effects of Acacia extract concentration on oviposition, three concentrations of 250, 500, and 1000 ppm in transparent glass beakers (6.5 cm high and 4.5 cm diameter, volume 200 ml) were prepared and exactly, 75 Cx.pipiens females were tested.After taking the blood meal, the number of egg rafts in the three cups was monitored for four to five days.The experiment was repeated three times with untreated control groups (dechlorinated tap water only).The hatching rate of mosquito eggs was also determined according to Zheng et al. 49 , where all egg rafts were examined under binocular microscope.Hatch rate was calculated after 72 h by dividing the number of eggs observed and considered to be hatched by the total number of eggs present on the paper.

Efficacy of selected plant extracts against non-target predators
Acacia methanol and aqueous extracts were tested against Gambusia affinis and Sphaerodema urinator and, two of the among field predators that live in mosquito's places.After catching, they were placed in plastic bags partially filled with water and transported to the lab.24 h, predators were fasted prior to the bioassay.They were then treated with LC 50 of Acacia extract, 3 predators per 1 l of dechlorinated water inside an enamel plate (35 × 25 × 15 cm) with 300 larvae.Three replicates were carried with controls.

GC/MS analysis
For the biochemical analyses, Thermo Scientific Trace GC Ultra/ISQ Single Quadrupole MS, TG-5MS fused silica capillary columns were utilized, with thicknesses of 0.1 mm, 0.251 mm, and 30 m.It was done with a 70 eV ionization energy electronic ionizer.Helium gas was employed as a carrier gas (flow rate: 1 ml/min).The MS transmission line and injector were both set to 280 °C.Preheating the oven to 35 °C, it was then raised to 150 °C at a rate of 7 °C per minute, 270 °C at a rate of 5 °C per minute (with a two-minute break in between), and finally 310 °C at a rate of 3.5 °C per minute (continual for 10 min).To investigate the quantification of every component found, a relative peak area was utilized.The substances were at least partially identified by matching their retention periods and mass spectra to NIST and Willy Library data from the GC/MS instrument.The aggregate spectrum of user-generated reference libraries was used for identification.Single-ion chromatographic reconstructions were carried out to assess peak uniformity.When possible, co-chromatographic analysis of reference chemicals was employed to validate GC retention durations 50 .

Fatty acid binding protein inhibition assay
Th inhibition assay of Fatty acid binding protein (FABP4) was determined according to Zimmerman and Veerkamp 51 .The solid aqueous and methanolic extracts were dissolved in DMSO to make the required serial dilution which has no detrimental effect on the fatty acid binding protein.FABP assay buffer was prepared via dilution of the (10 × buffer) by taking 2 ml and 18 ml HPLC-grade water.Followed by preparation of FABP assay www.nature.com/scientificreports/detection reagent, FABP4 assay protein, arachidonic acid and inhibitor/ligand concentrations according to Cayman chemical kit recommendations.In 96 well plat, 40 µl of FABP assay buffer to each well followed by adding of 25 µl of the detection reagent and 10 µl of the inhibitor/ligand dilution.Then the plates were covered and incubated for 10 min and the fluorescence measured at 475 nm after excitation at 370 nm using a plate reader.

Source of the objective protein
The binding affinity of the recognized active ingredients from both aqueous and alcoholic acacia extract against lm-FABP binding site were examined using one of the theoretical approaches in molecular docking, MOE tool, to decide whatever the active ingredients could make successive binding with the target protein and accordingly suggesting the mode of action.There is not a three-dimensional structure of fatty acid binding protein owing to Cx. pipiens in the protein data bank, hence the well-known crystal three-dimensional structure of the Fatty acid binding protein from locust flight muscle in complex with oleate lm-FABP(PDB code:2FLJ) 43 was utilized for this purpose and downloaded from protein data bank (https:// www.rcsb.org/ struc ture/ 2FLJ) as PDB format, water molecules were excluded in addition to any heteroatom subtracted and only chain a constrained.

Energy minimization
The most abundant and activated ingredients from both aqueous and alcoholic acacia extracts were automatically identified by the GC/MS.A database set of twenty candidates of the active ingredients were chosen to perform this study, were drawn using CAMBRIDGESOFT CHEMOFFICE 2015 Professional 15.0.0 software after recalling their smiles from PubChem database.The drawn structures were saved as Mol format after fulfillment energy minimization step using the defaults' function Amber12: EHT forcefield till gradient convergence of 0.01 kcal/ mol.Energy minimization step in addition to molecular simulation were done using the software Molecular Operating Environment MOE_2015.10,which installed by 64-bit operating system [Intel (R) Core (TM) i5-2400 CPU @ 2.40 GHz, 8 GB RAM] system.

Docking procedure
After uploading lm-FABP (PDB code: 2FLJ), both twenty candidate ligands and target protein were prepared, the reference drug (oleate) was assorted in green color to be distinguished easily.The binding site was recognized automatically from the surfaces and maps option and accordingly the co-crystallized ligand's binding site, pocket site, was separated and saved as MDB file.After assorting the twenty candidates in energy-minimized in new database, the docking step was done from "compute" option using the defaults of "Rotate bonds" choice to permits flexible-ligand rigid receptor docking to be fulfilled.Additionally, the scoring energy function was changed to be London G with triangle matcher replacement were set.A "fifteen conformers" was re-adjusted instead of the "thirty" automatic option conformers of the best score energy done by the ligand.One of top five conformers of best energetically favored ligand-protein docking was chosen to represent the ligand docking and showed two-and three-dimensional receptor interactions 52 .Docking results of all the tested twenty candidates were assorted into one table, considering the preference of number of interactions, scoring energy (kcal/mol), RMSD (Å), and the bond length (Å).Three-and two-dimensional docking interaction were captured, and the green color was used to indicate the ligand under docking investigation to be easily distinguished, intermolecular hydrogen bonding and π-π staking (aromatic) were labelled in magenta and yellow color, respectively, meanwhile the loops, helical, etc. of protein structures were colored automatically by the software and some renderings set up are made for best manifestation show.

Fatty acid binding protein activity test (FABP4)
Fatty acid binding protein (FABPs) are group of intracellular liquid binding protein (iLBP) usually binds to intracellular hydrophobic ligands and shielding them throughout cellular compartments.The activity of FABP4 caused by both aqueous and methanolic acacia extracts treatment were observed to be diminished and the minimal inhibitory concentration caused by methanolic extract 0.681 µg/ml meanwhile aqueous extract 2.311 µg/ml against the positive control, orlistat, 0.535 µg/ml.Studies conducted in FABP4 levels in mice have proven that the down-fluctuations of such proteins are related to many metabolic diseases 53,54 .With crucial role future clinical treatments.Various effective FABP4 inhibitors were developed but unfortunately, none of them approved or even entered clinical trials 55 .BMS-309403, FABP4-ap2, is a very strong, selective adipocyte fatty acid binding protein inhibitor with minimal inhibitory concentration ki = 2 nM (0.9 µg/l), 250(118 µg/l), and 350(165 µg/l) nM for FABP4, FABP3, and FABP5, respectively 56 .Comparatively, Methanolic acacia extract exhibited good inhibition ability with ki = 0.68 mg/l and being from natural source that may encourage its safety attitude.

Statistical analysis
The data were analyzed using the software SPSS V23 (IBM, USA) to perform Probit analyses to compute lethal concentration (LC) values and the one-way analysis of variance (ANOVA) (Post Hoc/Turkey's HSD test).The significance threshold was established at P < 0.05.  1) and the corresponding LC 50 (50%, median lethal concentration) = 200.50ppm (Table 2), while the corresponding values for aqueous extracts were 98 (MO%) and the LC 50 values = 256.40ppm.

Mosquito larvicidal activity
The larval mortality was maximum after 48 h of PT, while the mortality reaching 100% at 1500 ppm in methanol and aqueous extracts.In terms of fatal concentrations, A. nilotica methanol extracts revealed to be the most effective against Cx.pipiens larvae (LC 50 = 140.13ppm), followed by aqueous extracts (LC 50 = 172.68ppm).The larval mortality rate for the traditional positive control (temephos) was 73.3 and 100% at 24 and 48 h post treatment, respectively in aqueous and Methanol extracts.

Effect of Acacia extract concentration on eggs hatching rate
We tested the oviposition and hatchability rate of eggs in the A. nilotica solution.Exactly 25 Cx.pipiens females were used for the test and mixed with 30 of the mosquito males, and consequently, 122 egg rafts were counted, and the detailed number of laid eggs and hatching rate were summarized in Table 3 and Fig. 1.At a higher concentration of A. nilotica extract (1000 ppm), about 65 egg rafts were laid and reached a hatching rate of 24.4%.
Table 1.Efficacy of Acacia nilotica methanol and aqueous extracts on Cx. pipiens mortality, 24 and 48 h posttreatment.Numbers of the same column followed by the same small letter (a-g) are not significantly (P > 0.05) different (one-way ANOVA, Duncan's MRT).* Temephos insecticide at 0.25 ppm.

The Efficacy of plant extracts against non-target predators
The effects of the Acacia plant extracts on non-target insects (G.affinis and S. urinator) were tested.They were treated with LC 90 values of A. nilotica aqueous (847.23), and A. nilotica methanol (711.24ppm), respectively, against these predators (Table 4).The mean predation for A. nilotica methanol and aqueous with chosen predators did not differ significantly (P > 0.05).According to the data, mosquito fish, G. affinis (78.67, 80.67%) and S. urinator 26, 25.67%) bug were not affected in both methanol and aqueous plant extracts (Table 4).

FABP4 enzyme assay evaluation
The FABP4 enzyme inhibition assay for both methanolic and aqueous of acacia plant extract were estimated and orlistat was used as positive control and their half-maximal inhibitory concentration (IC 50 ) 0.681, 2.311 and 0.535 µg/ml for the methanolic extract, aqueous extract and the lipase inhibitor, orlistat, respectively (Fig. 3).

Docking study
The target protein Lm-FABP, PDB:2FLJ as fatty acid binding protein in  as showen in Fig. S5 (Fig. 7).

Discussion
Plant extracts usually combines of various safe, and biodegradable essential oils (EOs) active ingredients may use as pests and disease control that killing harmful pests effectively.Merely 5% of pesticides worldwide are biopesticides, despite their advantageous effects as insecticides 57 .However, due to their special qualities that promote their application, such as their non-toxicity to the environment, biopesticides are growing quickly and are predicted to surpass chemical pesticides in the near future at an average annual growth rate of 9-20% [58][59][60] .
All the Acacia plant extracts that were evaluated in this study demonstrated a high level of insecticidal efficacy against mosquito larvae.Acacia methanol extracts killed mosquito larvae at 100%, 24 and 48 h post-treatment, and they were more effective than Aqueous extract.Our findings corroborated those of Baeshen and Baz 35 research on the effects of extracts from Salix safsafs, Eucalyptus camaldulensis, and A. nilotica on Cx. pipiens larvae.They discovered that the extract from A. nilotica reduced the number of female eggs and killed mosquito larvae (100% mortality).Similarly, A. nilotica acetone leaf extracts were found to be acutely toxic at 212.1 mg/l and chronically toxic at 144.2 mg/l by Zaitoun et al. 61 , also who referred to the ability of A. nilotica extracts on inhibit adult emergence and egg hatchability.
Many multifunctional active ingredients have apparently been discovered in the seeds and leaves of A. nilotica 62,63 .Consequently, extracts from A. nilotica's fruits and leaves have demonstrated potential as fungicides, bactericides, insecticides, and molluscicides 62,64 .Vivekanandhan et al. 65 assessed the bio efficacy of A. nilotica seed essential oil and seed pod solvent extracts against three significant mosquito species 24 h after treatment.
In addition to their effectiveness on mosquito larvae, A. nilotica's methanol and acetone bark extracts were examined using an artificial diet bioassay for their potential to inhibit the growth of melon fly (Bactrocera cucurbitae) larvae in their first, second, and third instars.Also, pupation and emergence percentages were reported to be markedly decreased 66 .Studies have shown that extracts from A. nilotica can be effective in killing mosquito larvae.These extracts can disrupt the larvae's life cycle and prevent them from developing into adult mosquitoes 14,67,68 .In addition to being highly effective against other insects 65,69 and microbes 63,70 .
A. nilotica extracts have been shown to be safe in research studies, meaning they can be used in aquatic environments where mosquitoes live.To ensure they are safe for existing organisms and to see what would happen if these extracts were used extensively, we evaluated these extracts on some non-target aquatic organisms common in mosquito larval habitats.It was found that there were no significant differences (P > 0.05) when applying aqueous extracts to predators, whether with aqueous or ethanol extracts.
Three of the non-target aquatic biocontrol agents were less hurt after being treated with A. caesia extracts and Ag NPs than mosquito larvae.The A. caesia LC 50 ranged from 684 to 2245 μg/ml.Overall, our study shows that A. caesia has only a moderate effect on non-target aquatic biocontrol agents like A. bouvieri, D. indicus, and G. affinis fishes 71 .Specifically, the literature articles discussing the effects of the extract from the Acacia plant on fish or non-target organisms are rarely.However, generally speaking, a large number of literary articles support the idea that medicinal plants are not toxic to fish or non-target organisms.Promsiri et al. 72 found that the guppy fish (Poecilia reticulata) was a common non-target organism found in Ae. aegypti habitats at levels that were effective at killing mosquito larvae.This is an example of this.They discovered that extracts from 112 different medicinal plant species showed no or very low toxicity to guppy fish.In another study that conducted the toxicological effect of Plumbago zeylanica and Cestrum nocturnum plant extracts against the fish species P. reticulata, which are the most widespread non-target organisms in A. aegypti environments, almost no significant harm was reported at doses of LC 50 and LC 90 73 .As an attractive point, the descending concentration gradient leads to a descending rate of oviposition and hatching rates.At high concentrations of A. nilotica extract, the oviposition rate recorded its maximum value, and more than 50% of the total eggs were laid.Similarly, oviposition attitude was observed at moderate concentration, and less than 31% of the total eggs were counted.In agreement, the use of a very low concentration of A. nilotica showed a noticeable downfall of the number of eggs laid to less than 13% and 5% of the total eggs laid for the concentrations 250 ppm and zero (control without extract), respectively, confirming the direct relationship and oviposition rate.Moreover, the hatching rate is inconsistent with the oviposition rate.The hatchability rates were affected by the concentration gradient, and at zero concentration, the hatching ability was raised to 95% of the eggs laid at this concentration, whereas the higher concentration revealed a lower hatchability rate, confirming the inverse relationship between the concentration and the hatching rate.Based on the experimental data, the concentration of A. nilotica extract is directly proportional to oviposition rate and inversely proportional to hatching ability, which may introduce a reasonable interpretation for the dual benefit of acacia.Through what some active ingredients do to attract insects 74,75 , perhaps through extracting odorant chemical code, at the same time, the rest of the active ingredients help kill the larvae and reduce the chances of eggs hatching 76 .Bioactive plant chemicals, bacteria, fungus, viruses, and protozoa are natural sources of bioinsecticides, as are pheromones and microorganisms.According to their source, the four main classes of bioinsecticides are phytochemicals, plant-incorporated protectants, microbiological pesticides, and pheromones 77 .They have been successfully used to manage and control pests 78 .Because they are more potent at very low doses, being biodegradable, less toxic, and target-specific than synthetic compounds, they are superior to them.
Plants are biological factories that generate a wide range of compounds referred as secondary metabolites.The main pharmacological effects of all medicinal plants are attributable to those secondary metabolites, which include alkaloids, carbohydrates, flavonoids, saponins, tannins, and terpenoids.Several different types of bioactive compounds that are isolated from plants or added to botanical pesticides may have negative effects on pests or other species that eat them or come into contact with them 79 .
The study examined the organic compounds present in the methanol and aqueous extracts of A. nilotica.The results indicated that the methanol extract had a greater variety of organic compounds, including terpenes, steroids, polyethylene glycol, and fatty acid compounds, whereas the aqueous extract had a higher concentration of fatty acid contaminants.The extracting solvents' polarity had an impact on the antioxidant qualities, phytochemical content, and extraction yield 80 .Different extraction yields were obtained with different solvents.This is because the plant materials include high quantities of polar chemicals that are soluble in high polarity solvents, which might lead to a considerable fluctuation in the quantity of bioactive compounds in the extract due to changes in the polarity of the solvents.The findings showed that acetone contained more phytochemicals than aqueous extract, including phenols, terpenes, flavonoids, alkanes, and ketone 81 .
We find that depending on the type of solvent used in extraction and the presence of the Acacia plant in a specific area with specific climate, a clear variation appears in the secondary compounds.According to Edriss et al. 68 , the A. nilotica water and ethanol extracts revealed a broad spectrum of secondary metabolites, primarily alkaloids, saponins, flavones, and tannins, but the petroleum ether extracts were primarily composed of triterpenes and sterols.Aqueous and alcohol extracts of the plant A. nilotica were subjected to phytochemical screening.Acacia nilotica fruit showed the existence of phenol, terpenoid, flavonoid, reducing sugar, steroid, and flavonoids 82 .
A. nilotica contains various bioactive compounds, including tannins, alkaloids, and flavonoids.These compounds are believed to be responsible for its larvicidal activity, where in the Table 4 there are some of the active ingredients that are used in medical treatments, such as Methoxyphenyl piperazine (Piperazine).
Piperazine and its derivatives have attained a unique position as imperative pharmacophores in several therapeutic areas.Researchers have always been interested in using piperazine molecules as a building block to make new drug molecules that have a lot of different biological activities 83 because of their flexible structure.Neophytadiene is a good analgesic, antipyretic, anti-inflammatory, antimicrobial, and antioxidant compound 84 .
The mission of carrying lipids and poorly soluble molecules such as retinoids, fatty acids and bile acids in addition to all hydrophobic constituents transported by the cytosol for variant purposes of storage or even utilization, is one of the intracellular binding proteins's roles in the living biology.Such proteins are a family of lowmolecular weight and single chain polypeptides binds to the hydrophobic components, from different origins, forming a matrix of complex capable of carrying poorly soluble compartments into cytosol or any organelles in the cell, this complex keep the sensitive poorly soluble components shielded from decomposition.Fatty acid binding proteins, FABPs, aids to synthesize phospholipids, lipid metabolism and mitochondrial beta oxidation furthermore a large number of functional roles of FABPs have been proposed based on physiological disorder in both animal and insect kingdoms 85 .FABPs usually expressed largely in both vertebrates and invertebrate 86 .FABPs classified into main categories in mammels constrained epidermal (E-FABP), intestinal (I-FABP), brain (B-FABP), haert (H-FABP), myelin (m-FABP), testis (T-FABP), retina (FABP12) and liver FABPs 87 .Majority of FABP proteins share high amino acid sequence similarities more than 70% however, their three-dimensional structure was highly constrained β-barrel structure with ligand binding cavity [88][89][90] .
The first FABP discovered was desert flight muscle locust of Schistocerca gregaria and Haunerland et al. 91 followed by two abundant MFB1 and MFB2 were isolated from the midgut of Manduca sext larvae 92 .FABPs from different and variant insects heavily affects insect physiological metabolism by regulating intracellular fatty acids, it regulates sleep and long term memory consolidation, affects lipid accumulation in addition to their role in nutritional exploitation and caste division of many insects 93 .The change in FABPs chemistry will promote extensive physiological and behavioral attitude of insects.Hereon we tried to introduce a rationalized www.nature.com/scientificreports/mode of action describing the activity of acacia extract, and how it affects Cx.Pipiens, taking the advantages of the great structure similarity of their fatty acid binding proteins (FABPs) for most insects.We used lm-FABP (PDB code:2FLJ) as model to evaluate each single component of the activated ingredients in acacia extract and how it binds to the target FABP.Qualitatively, the docking results of most of the activate ingredients detected by the GC/MS were found to be close to the positive control, OLA, Due to the structure similarity between most of detected compounds and OLA.The results divided into four main categories the first one is some of tested compound revealed the same number of bindings with great similarity of amino acids residues like compound 1, methoxyphenyl piperazine, and compound 4, hexadecenoic acid, each one on them revealed three dipole-dipole interactions.The second category was recognized with candidates bonded with two hydrogen bonds like compounds (z,z,z)-9,12,15octadecatrienoic acid [6]; 7,10,13-eicosatrienoic acid, methyl ester [8]; 9,12-Octadecadienoic acid (z,z)-, methyl ester [16]; Stearic acid methyl ester [17]; Cis-11-eicosenoic acid [18] and Ethyl iso-allocholate [19].The nature of hydrogen bond shares the same properties of length, they have comparable length, and with the same amino acids' residues e.g., the ester group in compound 8 interacted with Try130 and Arg128 amino acids, this is quite similar to the reference drug OLA.
Similarly, compound 18 and 19 revealed the same number of interactions.As shown in Fig. 8 (supplementary file) compound 18 has relatively high and not preferable RMSD.Such value determines whatever the tested ligand could simulates the co-crystallized ligand or not.Energetically the accepted value of RMSD should not exceed 1.7 and higher values are energetically not favored 41,42 .On the other side, a group of six candidates of 10, 11, 12, 13, 15 and 20 showed a smaller number of bonds however some of these compounds interacted with the same residual amino acid as the reference drug e.g., compound 10.In addition to RMSD values, the scoring energy is a very beneficial physical property and all the selected poses revealed negatively charged scoring energy.The larger the negative value of the scoring energy becomes, the more stability it indicates, and for this compounds 8 and 17 have the lowest scoring energy of -7.4504 and -7.3073 kcal/mol, respectively.
From the docking results, most of the tested structures such as 2, 4, 5, 6, 8, 15, 16 17 and 18, showed at least one interaction resemble to the OLA.The most effective factor is the structure of that compounds and their structure-resemblance with oleate (OLA) co-crystallized ligand.Such compounds have long hydrocarbon chain prolonged from 14 to 20 carbon atoms (Fig. 9) and the reference drug consist of exactly 14 carbon backbone.Such resemblance makes the size of tested ligand fitting to the protein receptor pocket and that interprets the low RMSDs which strengthen the idea of compatibility.Additionally, the carboxylic (COOH) group and /or ester group (COOMe) presented in the tested compounds structurally agreed with the carboxylate (COO -) terminated the OLA.
Extending carbon backbone more over e.g., squalene, even if it contains active part like multiple double bonds, but the size of the structure becomes non-compatible to the receptor pocket.Replacing the long chain hydrocarbon with polycyclic moieties as showed in Fig. 10 such as compounds 7 and 13 did not achieve the same purpose of binding ability.

Conclusion
Molecular hybridization and docking simulation were used to introduce a convenient mode of action and to suggest how is acacia extracts affects Cx. pipiens via studying the binding compatibility of each single component detected by the GC/MS to the FABP, as one the effective proteins the invasively affect not only Culex but all vertebrate and invertebrates.Three components manifested unique binding ability like OLA co-crystallized ligand.Some other candidates presented exceptionally good binding abilities with higher chances of replacing OLA.The activity prediction by simulation keeps the ways smoothed for the researchers to continue exploring the potential of A. nilotica and its extracts as a natural and sustainable means of controlling mosquito populations and reducing the spread of mosquito-borne diseases.However, the effectiveness of these extracts may vary depending on factors such as concentration, application method, and local environmental conditions, so further research and testing are necessary to refine and optimize their use.

Fig. 4 Figure 1 .
Figure 1.Efficacy of A. nilotica plant extracts concentration on the oviposition and hatching rates of female Cx.pipiens.

Figure 2 .
Figure 2. The TIC chromatograms of Acacia nilotica methanol (a) and aqueous extracts (b) chemical constituents detected by GC-MS.

Figure 3 .
Figure 3. FABP4 enzyme assay inhibition of the tested acacia extracts.

Figure 4 .
Figure 4. Three-dimensional structure of 2flj and its active pocket.

Figure 10 .
Figure 10.chemical structure of the most active and abundant activate ingredients of polycyclic components of acacia extract.

Table 5 .
The major chemical constituents of Acacia nilotica methanol extracts.

Table 6 .
The major chemical constituents of Acacia nilotica aqueous extracts.

Table 7 .
Docking results of the most abundant active ingredients in acacia aqueous and methanol extract to the vicinity of Lm-FABP, PDB:2FLJ fatty acid binding protein.